Method for production of fermentable sugars from biomass

ABSTRACT

A process for production of fermentable sugars from biomass using multi-enzyme multi-step system is provided herein. The process disclosed in the present invention provides high yielded sugars in less time period. The multi-enzyme system disclosed in the present invention converts celluloses, hemicelluloses and/or mixture thereof to fermentable sugar with higher efficiency and better economics than the process known in the prior art. Cellulose and hemicelluloses fractions derived from natural sources such as any lignocellulosic biomass are saccharified in a shortened time with higher conversion rates of intermediates with modified enzymatic compositions/groups of the Multi-enzyme system to enhance the rate thus providing an economical cellulose and hemicellulose saccharification process.

This application claims priority from provisional applications number1299/MUM/2009 filed on May 26, 2009 and 1314/MUM/2009 filed on May 29,2009.

FIELD OF INVENTION

The present invention relates to the field of production of fermentablesugars from biomass for production of biofuel and other by-products.

BACKGROUND OF INVENTION

Lignin and two polysaccharides hemicellulose and cellulose form thethree major components of plants physiology and are collectively calledas lignocellulose. Of these three, cellulose and hemicellulose arebasically polymers of sugar monomers like glucose, xylose, galactose,arabinose etc. Therefore, cellulose and hemicellulose derived from plantresidues, if hydrolyzed to monomeric sugars, can form a useful andabundant renewable source of raw material for a variety of usefulchemicals and biochemicals. Conversion of this generally tightlycompacted composite lignocellulosic material to sugar is accomplished bya composite process known as hydrolysis and saccharification. Worldwideresearch on saccharification processes for the conversion oflignocelluloses to sugars has followed three major approaches. First ischemical hydrolysis, the second is thermal hydrolysis and the third isenzymatic hydrolysis.

In a general chemical hydrolysis process, hemicellulose is separated inthe first step from the lignocellulose composite material by the actionof an acid or alkali. The plant material/mass is mixed with a dilutesolution of an acid or alkali and then heated. This process releases and“hydrolyzes” the hemicellulose. Hydrolysis of hemicellulose producespentose sugars (C5 sugars) as well as some hexose sugars (C6 sugars).The second step is a higher temperature acid hydrolysis process thathydrolyzes the plant material cellulose, producing almost solely C6(hexose) sugars, and lignin. The C6 sugars, when separated substantiallyfrom lignin, are readily fermentable, and the recovered lignin can beused for process heat or making other products.

Two stage acid hydrolysis processes have been used for many years.However, it is now known that the acid processes also produce chemicalsother than sugars that not only represent a process loss but also leadto problems later in the use of the sugars in downstream processes likefermentation to useful products like lactic acid, alcohols, organicacids etc. Another major problem with these systems has been that theacid must either be recovered for re-use or it must be neutralizedthrough the use of lime in order to mitigate effluent and pollutionproblems.

Autothermal processes on the other hand do not make use of any chemicalsand thus are cleaner processes. High temperatures and short exposureslike used in Steam Explosion processes, results in breakdown of thelignocellulosic biomass into monosugars and hydrolyzed lignin. However,such processes suffer from the drawbacks of lower sugar yields,formation of unwanted side-products that are inhibitory to downstreamprocesses, and are energy intensive.

Use of enzymes, generally preceded by some or the other mildpretreatment steps, provides much cleaner and low energy process forcellulose and hemicellulose hydrolysis and saccharification and finallyprovides better quality end products i.e. sugars in higher yields.

Several enzymes are known to specifically, or non-specifically,hydrolyze plant cell wall polysaccharides. Such enzymes derived fromculture filtrates of microorganisms have found large scale applicationsfor hydrolysis of cell wall components (Reese, E. T. et al, Can. J.Microbiol. 19, 1973, 1065-1074). Microorganisms produce numerousproteins, and some also produce cellulose and/or hemicellulose splittingenzymes. Most reports and technologies make use of these catalyticenzymes in free soluble form that cannot be recovered for reuse.Further, often the substrates namely cellulosic and/or hemicellulosicpolymers and products of hydrolysis thereof, have tendencies to‘inhibit, the enzymes’ actions. Such a use of these enzymes makes themless attractive for use on a commercial scale or makes the use of theenzymes more expensive than often desired. Therefore, for reasons ofcost, the amount of enzymes used per unit weight of cellulose and/orhemicellulose hydrolyzed is often kept to a minimum, which in turnreduces the rate of hydrolysis reactions and increases the reactiontimes.

For the reasons stated above, and for other reasons stated below whichwill become apparent to those skilled in the art upon reading andunderstanding the present specification, there is a significant need inthe art for systems and methods that provide for improved biomassconversion to sugars in a cost-effective manner. Shortcomings of theenzyme process can be alleviated making it the obvious choice for newprocess development.

Cellulose and hemicellulose are the first and second most abundantpolysaccharides in nature. Cellulose represents anywhere from 30 to 60%while hemicelluloses represent about 20-35% of lignocellulosic biomass(LBM) such as corn fiber, corn stover, wheat straw, rice straw, andsugarcane bagasse. While cellulose is an almost homogeneous polymercomprised of several hundreds to thousands D-glucose units linkedthrough 1,4 β-glycosidic linkages, hemicelluloses are heterogeneouspolymers of pentoses (xylose, arabinose), hexoses (mannose, glucose,galactose), and sugar acids. Hardwood hemicelluloses contain mostlyxylans, whereas softwood hemicelluloses contain mostly glucomannans.Xylans of most plant materials are thus heteropolysaccharides withhomopolymeric backbone chains of 1,4-linked β-D-xylopyranose units.Besides xylose, xylans may also contain arabinose; glucuronic acid orits 4-O-methyl ether; and acetic, ferulic, and p-coumaric acids. Thefrequency and composition of branches are dependent on the source ofxylan while the backbone consists of O-acetyl, α-arabinofuranosyl,α-1,2-linked glucuronic or 4-O-methylglucuronic acid substituents.

For both cellulose and hemicellulose components to be efficientlyconverted to their monosugar components these must first be extractedfrom the lignocellulosic complex. Enzymatic saccharification of thesetwo components using cellulases and hemicellulases is the preferredmethod due to rapid action of the enzyme, and negligible substrate lossand side product generation. Both cellulose and hemicellulose in intactLBM however, are not accessible to enzymatic hydrolysis. And thereforepretreatment of the LBM to render these amenable to enzyme action ismandatory (Himmel, M. E. et al, 2007; Bothast and Saha, 1997). Whilecellulose, though a homopolymer, is a far more bulkier, crystalline andcompact molecule, the structure of hemicellulose is more complex as itcomprises of pentoses, some hexoses and side chain groups such as acetyland uronic acids. Thus, enzymatic hydrolytic action for both celluloseand hemicellulose requires combined action of more than one enzyme. Forcellulose hydrolysis the crystal structure of cellulose needs to bepartially or wholly rendered amorphous after which a mixture of exo andendo cellulases is required for conversion of the polymeric cellulose tomuch smaller oligomeric molecules. On the other hand, in case ofhemicellulose, the presence of side chain groups hampers the action ofmajor backbone depolymerizing enzymes i.e. exo and endo xylanases, andmannanases. To address this problem accessory enzymes such asα-L-arabinofuranosidase, α-glucuronidase, acetylxylan esterase, ferulicacid esterase, and p-coumaric acid esterase which have the ability tohydrolyze the side chains have to be present with the majorhemicellulases to achieve complete degradation of hemicellulose toobtain high yields of monosaccharide sugars (Biely and Tenkanen, 1998).

As a result of such scenario, cellulase and hemicellulase preparationsused for depolymerizing or hydrolyzing cellulose and hemicellulose,respectively, contain a myriad of major and minor enzymes that all acttogether.

However, on the other hand, it is now well recognized that, the startingand intermediate substrates occurring during the sequential butcomplicated process of polymer hydrolysis, tend to act as partial orcomplete inhibitors of the enzymes present in the mixture preparationsused (Beguin P et. al, (1994), FEMS Microbiological Review, 13, 25-58and Ven H Tilbeurgh et al, Studies of the cellulytic system ofTrichoderma reesei QM 94014 (1989), European journal of Biochemistry,189, 553-559). As a result of this fact, and the fact that one may notwant to use excessive quantities of enzymes for cost reasons, theenzymatic saccharification processes for both cellulose andhemicellulose are long duration reactions requiring 24 to 48, and oftenmore, hours for completion. It has long been accepted that enzymes aretruly efficient catalysts. However, since derived from biologicalsources and purified, at least partially, and on account of theirinherently complex, fragile and sensitive nature, enzymes are expensiveand unstable. This has put severe limitations on the spectrum and scaleof applications of enzymes in industry (F. Dourado et al, 2002, Journalof Biotechnology, 99, 121-131). Several methods have been devised torender the enzymes stable and less expensive for use for productionscale applications. Thus, new enzymes, including cellulases andhemicellulases, have been developed and manufactured such that they arestable to wide temperature, pH and other harsh conditions like presenceof inhibitors (Khare and Gupta, 1988, Applied Biochemistry andBiotechnology, 16, 1-15, Busto et al, Bioresource Technology, 1997, 60,27-33). However, despite these efforts, these enzymes today contributesignificantly to the cost of conversion of cellulose and hemicelluloseto simple sugars.

One way of reducing enzyme cost is to use the enzymes in immobilizedform, or in a form, or way, that permits reuse of enzymes over manycycles, or over extended periods of time. Thus, in a reusable form orway, the enzymes are retained in the reactor, while the substrate/s andproduct/s flow in and out, in batch or continuous fashion. However, useof an enzyme in immobilized form on a solid support, requires thatreactants (or substrates) and products are in soluble form to facilitatethe reaction. Further, when using enzymes for reactions involvingpolymeric reactants and products (like cellulose and hemicellulose), theaccessibility of the enzymes in the pores of the immobilization supportbecomes rate limiting and the reactions become too slow to be ofpractical use (Woodward J. 1989, Journal of biotechnology, 11, 299-311).This, and the fact that cellulose is an insoluble solid andhemicelluloses are polymeric with low solubility in water as well, hasprevented use of cellulases and hemicellulases in recyclable and/orimmobilized forms.

U.S. Pat. No. 4,200,692 discloses a process for the production of xyloseby enzymatic hydrolysis of xylan wherein the enzymes are immobilisedseparately but incubated together and, the xylan solution is broken toxylobiose and xylose and acid sugars. After 4 hours total hydrolysis toxylose and 4-O-methylglucuronic acid is claimed. US2008/065433 disclosesa process for obtaining fuel ethanol by using agricultural andagroindustrial waste materials composed of lignocellulose, andespecially sugar cane bagasse. The hemicellulose fraction is submittedto mild hydrolysis with sulphuric acid, and the solid material from thishydrolysis is submitted to a process of saccharification (enzymatichydrolysis) with simultaneous rapid alcoholic fermentation underconditions which allow a significant increase in conversion to alcoholin a greatly shortened time, approximately 8-32 hrs.

U.S. Pat. No. 6,423,145 discloses a modified dilute acid method ofhydrolyzing the cellulose and hemicellulose in lignocellulosic materialunder conditions to obtain higher overall fermentable sugar yields,comprising: impregnating a lignocellulosic feedstock with a mixture ofan amount of aqueous solution of a dilute acid catalyst and a metal saltcatalyst, loading the impregnated lignocellulosic feedstock into areactor and heating for a sufficient period of time to hydrolyzesubstantially all of the hemicellulose and greater than 45% of thecellulose to water soluble sugars; and recovering the water solublesugars.

US2009/098618 discloses a method for treating plant materials to releasefermentable sugars. Lignocellulosic materials are subjected to discrefining together with enzymatic hydrolysis to produce sugar richprocess stream that may subsequently be subjected to fermentation toproduce biofuels and chemicals.

U.S. Pat. No. 5,348,871 discloses a process for converting cellulosicmaterials, such as waste paper, into fuels and chemicals utilizingenzymatic hydrolysis of the major constituent of paper, cellulose. Wastepaper slurry is contacted by cellulase in an agitated hydrolyzer. Theglucose produced from hydrolyzer is fermented to ethanol in acontinuous, columnar, fluidized-bed bioreactor utilizing immobilizedmicroorganisms. The process disclosed in the patent requires ‘many hoursto days for acceptable yields’.

U.S. Pat. No. 5,637,502 discloses a batch process for convertingcellulosic materials into fuels and chemicals, such as sugars andethanol, utilizing enzymatic hydrolysis of cellulose. Waste paper slurryis contacted by cellulase in an agitated hydrolyzer. An attritor and acellobiase reactor are coupled to the agitated hydrolyzer to improvereaction efficiency. Additionally, microfiltration, ultrafiltration andreverse osmosis steps are included to further increase reactionefficiency and recycling of the enzymes. The resulting sugars areconverted to a dilute ethanol product in a fluidized-bed bioreactorutilizing a biocatalyst, such as microorganisms. The time of hydrolysisof paper cellulose is about 24 hours.

U.S. Pat. No. 227,162 discloses a method for lignocellulose conversionto sugar with improvements in yield and rate of sugar production byusing ionic liquid pretreatment. However, the time required for completebatch enzymatic hydrolysis is within 16 to 36 hours for two of therepresentative biomass samples—corn stover, poplar which is asubstantially longer period.

U.S. Pat. No. 5,932,452 discloses a process for the hydrolysis of ahemicellulose substrate containing xylo-oligomers, obtained from steamexploded plant biomass or enzymatically partially pre-hydrolyzed xylan,with an immobilized enzyme. This process however, has the pre-requisiteof producing partially hydrolyzed hemicellulose which in turn needs tobe obtained from plant biomass through suitable process such as steamexplosion. Steam explosion is a hydrothermal process and is known toproduce furfural derivatives that are known to affect both enzymaticconversion, and later fermentation efficiencies.

US2008/076159 discloses methods to produce enzymes or novel combinationsof enzymes, which provide a synergistic release of sugars frompre-treated plant biomass. However, the disclosed process does notreduce the saccharification period which is in the range of 24-72 hours.

EP2017349 discloses a method for the direct enzymatic treatment of rawpolymeric feedstock and separation of the resulting soluble components.However, there is no mention of recovery and reuse of the enzymes, andthe hydrolysis duration is also a prolonged one.

WO/2006/063467 discloses a continuous process system for enzymatichydrolysis of pre-treated cellulose which comprises introducing aqueousslurry of the pre-treated cellulosic feedstock at the bottom of avertical column hydrolysis reactor. Axial dispersion in the reactor islimited by avoiding mixing and maintaining an average slurry flowvelocity of about 0.1 to about 20 feet per hour, such that theundissolved solids flow upward at a rate slower than that of the liquid.Cellulase enzymes are added to the aqueous slurry before or during thestep of introducing. An aqueous stream comprising hydrolysis productsand unhydrolyzed solids is removed from the hydrolysis reactor and aftersolid separation the unhydrolyzed cellulose is recycled. Also providedare enzyme compositions which comprise cellulase enzymes and flocculentsfor use in the process. In addition, a kit comprising cellulase enzymesand flocculent is described that is said to provide exposure of theenzyme to the substrate. Although the cellulose conversion is better inthis case than batch reactor, the time required is 48 hours to 200 atrespective enzyme loading of 32 units/g cellulose to 5 units/gcellulose.

WO/2009/004950 discloses that monosaccharide and/or a water-solublepolysaccharide can be produced with a high degree of efficiency byhydrolyzing a cellulose-containing material with a sulfonate-containingcarbonaceous material. The used sulfonate-containing carbonaceousmaterial can be reactivated and reused by carbonization and sulfonation,without the need of separating the sulfonate-containing carbonaceousmaterial from the unreacted portion of the cellulose-containingmaterial. This method, which does not use any enzymes, enables to reducethe cost for hydrolysis, can reduce the amount of waste materials, andtherefore can contribute to the global environmental conservation.

The concept of enzymatic hydrolysis of cellulose and hemicelluloses isknown since long. As described above, most enzymatic hydrolysisprocesses in use, or reported are batch processes and take 12-48 hrs forcomplete saccharification. More often, the enzymatic processes remainincomplete resulting in high enzyme cost and slow reactions leading tolow throughputs and hence high capital investment in large reactors.While use of higher dosage of enzymes can increase the hydrolysis rate,the cost considerations limit the dosages. Further, dosages of enzymesin typical batch processes are higher than desired on account ofinhibitory effects of reaction substrates and products on the enzymes.For this reason, new efficient methods are needed for cellulose andhemicellulose saccharification which will require lower enzyme dosagesper kilo of cellulose and hemicellulose, not require high temperaturesand pressures, will not generate hazardous byproducts, will be less timeconsuming, and require less energy, thus making the process moreeconomically viable.

At the scale at which a biomass to sugars plant is expected to operate(typically 100 to 1000 tons biomass/day) large reaction times implyhumongous sized enzyme reactors exceeding several 100 KL capacities. Itis therefore necessary to speed up the reaction rates thereby increasingvolumetric throughputs.

OBJECTS OF THE INVENTION

One object of the present invention is to provide a multi-enzymemulti-step system for complete conversion of hemicellulose, cellulose,and/or a mixture thereof, obtained from any lignocellulosic materialincluding but not limited to agricultural residue, herbaceous material,forestry residue, municipal solid waste, pulp and paper mill residue,paper waste or any other source.

Another object of the invention is to develop an efficient process, interms of rate of the process and the amount of enzyme used per unitamount of sugars produced, for the enzyme catalyzed hydrolysis ofhemicellulose and cellulose, and or any mixture thereof to fermentablesugars, wherein the process is efficient in terms of enzyme cost as wellas be time efficient, and adaptable on industrial scale.

It is still further object of the present invention to provide themulti-enzyme system in two, or more, steps for effectivesaccharification or depolymerization of cellulose and hemicellulose tofermentable sugars.

Yet another objective of the invention is to provide a multi-enzymesystem that comprises at least two groups of enzymes, with selection ofspecific enzymes from specific groups, for two or more stepsaccharification, the groups being decided by the nature of the enzymesand as described later below.

Another objective of the invention is to provide further the group/s ofenzymes to act as accessory enzymes as well as auxiliary enzymes, andwhich can be added during the process, or along with the first group ofenzymes, or second group of enzymes, and or in both the groups of themulti-step process using multi-enzyme system.

It is still further object of the present invention to optimize each ofthe steps of cellulose and/or hemicellulose saccharification process,with respect to temperature, pressure, pH, solvent used, time of contactand other parameters to achieve more than 90%, or 95% or 98% conversionwithin a few hours

SUMMARY OF THE INVENTION

One of the aspect of the present invention provides a process ofproduction of fermentable sugars from hemicellulose and/or celluloseusing multi-step multi-enzyme system, wherein the process comprisestreating hemicellulose and/or cellulose with at least one enzyme offirst group of enzymes at a temperature ranging from 30° C. to 90° C. toobtain a first hydrolysate, and treating the hydrolysate obtained instep (a) with at least one enzyme from second group of enzymes to obtainthe fermentable sugars; wherein the first group and second group ofenzymes are capable of hydrolysing the hemicellulose and/or cellulose

Another aspect of the present invention provides a process of productionof fermentable sugars from biomass using multi-step multi-enzyme system,wherein the process comprises mixing biomass with 5% to 10% w/v alkalihaving pH in the range of 12-14 at a temperature ranging from 50° C. to200° C. under 1.0 to 20 bar pressure for 5 minutes to 2 hrs to obtain abiomass slurry; filtering the biomass slurry to obtain filtratecomprising hemicellulose; and residue comprising cellulose; treating thefiltrate with alcohol to obtain a precipitate containing hemicelluloses;washing the residue with water to remove residual alkali to obtaincellulose; washing the precipitate to obtain hemicelluloses; treatingthe hemicellulose and/or the cellulose thus obtained with at least oneenzyme of first group of enzymes at a temperature ranging from 30° C. to90° C. to obtain a first hydrolysate; and treating the hydrolysateobtained with at least one enzyme from second group of enzymes to obtainthe fermentable sugars; wherein the first group and second group ofenzymes are capable of hydrolysing the hemicellulose and/or cellulose

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “fermentable sugars” used herein refers to all those sugars,and their mixtures, that are water soluble and can be used as carbonsubstrates by microorganisms.

The term “Hydraulic Retention Time” (HRT) used herein refers to theaverage time the reactants spend in the reactor system and that isavailable for the reaction which herein is the hydrolysis ofhemicellulose and/or cellulose.

The terms “hemicelluloses” and “celluloses” as used herein respectivelyrefer to enzymatcially hydrolysable hemicelluloses and cellulosesderived from any lignocellulosic biomass.

The present invention relates to a multi-step method for production offermentable sugars using a multi-enzyme system with selectively chosenmulti-enzymes to convert hemicelluloses and/or celluloses to fermentablesugars with higher efficiency in terms of time and enzyme utilizationand hence better economics than known in the art. Cellulose andhemicelluloses derived from natural sources such as any lignocellulosicbiomass, are saccharified in a shortened time with higher conversionrates of intermediates with modified enzymatic compositions/groups ofthe multi-enzyme system to enhance the rate, and hence economics of thecellulose and hemicellulose saccharification process.

In one embodiment of the present invention there is providedhemicelluloses and celluloses which are rich fractions that are derivedfrom any lignocelluloseic biomass, and that are amenable to enzymatichydrolysis resulting in more than 90% hydrolysis to respectivemonosugars without any mechanical and/or chemical treatment within andduring the enzymatic hydrolysis.

Cellulose and hemicellulose fractions obtained from fractionation andpretreatment of biomass can be used for the process. Pure cellulose andhemicellulose from the similar or other sources can also be used. In thepresent invention, cellulose and/or hemicellulose are saccharified inmuch lower time than generally known due to higher conversion orreaction rates per unit volume of the reactor and the overall amount ofenzymes used.

The present invention of a multi-step multi-enzyme depolymerization orhydrolysis process system includes separate and sequential use of enzymecombinations that break down hemicelluloses and celluloses tofermentable sugars and oligosaccharides that can be further converted touseful products. In several possible combinations, the multi-stepprocess achieves conversion of complex carbohydrates like cellulose andhemicellulose into fermentable sugars, and which together are referredto herein as “saccharification”.

Owing to the complex structure of polymeric hemicelluloses andcelluloses, several different types of enzymes are needed for theirenzymatic degradation or modification. Enzymes in combinations ormixtures, can degrade the sugar polymers namely cellulose andhemicelluloses to simple or oligomeric sugars. Most available enzymesfor such processes are indeed combinations or mixtures of enzymesobtained from microorganisms, plants, or other organisms; andsynergistic enzyme mixtures comprising enzymes or multi-enzyme productsfrom different, or same, microorganisms, plants or other organisms orthe enzymes and mixtures thereof, and can be prepared indigenouslyand/or obtained commercially.

One of the embodiments of the invention relates to the enzymes that canbe used in the invention that are of microbial origin wherein themicroorganisms can be, but not limited to, genetically engineered, ornatural. These enzymes are, for the purpose of this disclosure, broadlyclassified into two groups as follows for the two classes cellulases andhemicellulases.

The First group of enzymes comprises of endo-glucanase, exo-glucanase,endo-xylanase, exo-xylanase, mannanase and galactanase from any knownsource. These enzymes belong to the exo-acting and endo-actinghydrolytic enzymes family, which is characterized by their ability tobreak down different polysaccharides to produce short-chainoligosaccharides. These enzymes are produced by fungi, bacteria, yeast,marine algae, protozoans, snails, crustaceans, insect, seeds, etc., butthe principal commercial sources are filamentous fungi like Aspergillusniger, Trichoderma reesei etc. Xylanases can be isolated frompsychrophilic microorganisms. Production of xylanases, β-mannanases,arabinanases, and pectinases can be, for example, done by using thethermophilic fungus Thermomyces lanuginosus. The mesophilic fungusTrichoderma harzianum strain T4 produces extracellular xylanase andmannanase activities when grown in the presence of oat (Avenasativa)-spelt xylan and wheat bran as the carbon sources respectively.Xylanase and Mannanase can be obtained from Streptomyces galbus NR.Sources of cellulases, for example, glucanases include fungi likeAspergillus niger, Trichoderma reesei, Pharochaete chyrosporium,Fusarium solani, Trichoderma konigii, Sclerotiom rolfsii, etc.; bacterialike Sporotrichum pruniosum, Arthrorhizopus sp., Clostridiumthermocellum, Ruminococcus albus, Streptomyces spp, etc.

The Second group of enzymes comprises of xylosidase, mannosidase andglucosidase. These enzymes belong to the glycosidase enzyme family,which break down the oligosaccharides that are released by exo-actingand endo-acting hydrolytic enzymes, into monomeric sugars. Xylosidaseand/or the enzymes from the same group as well as accessory enzymes aregenerally produced along with xylanase or the main enzyme. Similarly,glucosidase and/or the enzymes from the same group as well as accessoryenzymes are also generally produced along with glucanase/s or the mainenzyme. For example Piptoporus betulinus, a common wood-rotting fungus,produces endo-1,4-beta-glucanase (EG), endo-1,4-beta-xylanase,endo-1,4-beta-mannanase, 1,4-beta-glucosidase (BG), 1,4-beta-xylosidase,1,4-beta-mannosidase and cellobiohydrolase activities. The fungusproduces mainly beta-glucosidase and beta-mannosidase activity in itsfruit bodies, while higher activities of endoglucanase, endoxylanase andbeta-xylosidase are found in fungus-colonized wood. β-glucosidases forcellobiose and cellulose oligomers hydrolysis can be obtained frommicroorganisms like Piromyces sp, Fusarium oxysporium etc.

However xylosidase and its family enzymes can be generated and furtherpurified from some specific microorganism from crude extracts. Forexample, β-D-Xylosidase is produced in maximum yield from Humicolagrisea var. thermoidea. β-glucosidase and β-xylosidase can also beproduced from a yeast-like Aureobasidium sp. Few other examples includebacteria such as Agrobacterium tumefaciens C58, Bacillus haloduransC-125, Bacillus subtillis 168, bifidobacterium longum NCC2705,Caulobacter crescentus CB15, Clostridium acetobutylicum ATCC 824,Streptomyces coelicolor A3(2), Thermotoga maritima, Xanthomonasaxonopodis pv. Citri str. 306, Xanthomonas campestris pv. campestrisstr. ATCC 33913, Cellulomonas fimi, Cellvibrio japonicas, Geobacillusstearothermophilus T-6, Geobacillus stearothermophilus 21, Penicilliumwortmanni, and Bacillus pumilus.

The available and commercial preparations of both cellulases andhemicellulases from different sources are combinations, in differentproportions, of the various enzymes including the enzymes from the twogroups described above.

In the present invention, hemicellulose and cellulose, or any mixturethereof is saccharified in two or more steps involving enzymes from theabove two groups. The First step uses an enzyme preparation thatcontains at least one enzyme from first group, and may or may notcontain other enzymes from the same and second group. In the Secondstep, the enzyme preparation used contains at least one enzyme from thesecond group, and which may or may not contain one or more enzymes fromthe first group. Auxiliary enzymes such as amylases, proteases, lipases,glucuronidases etc. can be optionally added to both or one of the twosteps for enhanced rate of hydrolysis. Auxiliary enzyme(s) or auxiliaryenzyme mixture disclosed herein are defined as any enzyme(s) thatincrease or enhance the rate of saccharification of celluloses orhemicelluloses.

It is obvious that a person skilled in the art can produce enzymes ofthe two groups from any natural or genetically modified organism such asplant, bacteria, yeast or fungi.

In one of the embodiments of the invention, the enzymes of the twogroups are generally components of most enzyme preparations commerciallyavailable and obtained as fermentation products but these enzymes can besubjected to separation steps prior to use.

In one embodiment, the hemicellulose and cellulose can be obtained frombiomass using one or more techniques such as physical, chemical, orphysicochemical processes like, thermal treatment, hydrothermaltreatment, organosolv treatment, steam explosion treatment, limeimpregnation with steam explosion treatment, hydrogen peroxidetreatment, hydrogen peroxide & ozone treatment, acid treatment, diluteacid treatment, alkali treatment, heat treatment, or ammonia fibreexplosion treatment.

Biomass includes virgin biomass and/or non-virgin biomass such asagricultural biomass, forest waste, commercial organics, constructionand demolition debris, municipal solid waste, waste paper and yardwaste. Common forms of biomass include trees, shrubs and grasses, wheat,wheat straw, sugar cane bagasse, corn, corn stover, corn kernelincluding fiber from kernels, products and by-products from milling ofgrains such as corn, rye, oat bran, wheat and barley (including wetmilling and dry milling) as well as municipal solid waste, waste paperand yard waste. The biomass can also be or include, but is not limitedto, herbaceous material, agricultural residues, forestry residues,municipal solid wastes, waste paper, and pulp, and paper mill and oilmill residues.

Surprisingly, in the present invention, it was found that the hydrolysisof hemicelluloses and/or celluloses to the fermentable sugars carriedout using a multi-enzyme system in two steps increased the overall rateof reactions and therefore reduced the time of the process to producethe fermentable sugars. In particular, when at least one enzyme from thefirst group of enzymes, and at least one enzyme from the second group ofenzymes are added in stepwise manner then the saccharification timereduces 5 to 8 fold compared to the known processes. Contrary to this itwas found that when at least one enzyme from the first group of enzymes(glycanases/xylanases) is added along with at least one enzyme from thesecond group of enzymes (glucosidases/xylosidases) in the reactionmedium in the same single step, the initial rate of reaction is high.However, the reaction becomes slow after some time and the totalconversion takes more than 24 hrs at the levels of reported enzymedosages of about 10 enzyme units/g hemicellulose and/or cellulose. Oneunit of the enzyme is defined as the amount of enzyme that liberates onemicromole equivalent of glucose/minute/mL of reaction volume.

In the multienzyme multi-step reaction disclosed in the presentinvention, the enzymes utilized may be prepared by the methods wellknown in the art, or may be obtained commercially.

One of the embodiments of the invention is the first step wherein atleast one enzyme is specifically selected from the first group. Thusenzyme preparation used in the first step comprises at least one enzymederived/selected from group comprising of endo and exo cellulases and/orxylanases, and any mixtures thereof. Similarly, in the second step, atleast one enzyme is specifically selected from the second group ofenzymes comprising of xylosidase, mannosidase and/or glucosidase, andany mixtures thereof. The reason for such sequential selection ofspecific enzymes from specific groups, and using such enzymes or groupof enzymes, in a step wise manner is that the products of glycanases andxylanases interfere with, or inhibit the action of glucosidases andxylosidases, and such interference reduces the activity of the addedenzymes and slows down the overall rate of hydrolysis ordepolymerization reaction.

Further embodiment of the invention relates to overcoming of thelimitations of the traditional combined use of the first group and thesecond group enzymes, which results in interference/s or inhibition/s ofthe enzymes by the reactants and reaction products therebyslowing downof the reaction rate. The present invention discloses a process wherebythe action of the two groups of enzymes is separated thereby resultingin higher overall reaction rates.

In the preferred embodiment of the invention, at least one enzyme from agroup of enzymes mentioned above is added sequentially in each of themulti-steps to act on the separated hemicellulose or cellulose, or anymixture of hemicelluloses and celluloses, to convert them to fermentablesugars for the production of ethanol and/or other useful products.

Thus, in the first step, an enzyme mixture, comprising at least oneenzyme from the first group of enzymes, with or without one or moreaccessory or auxiliary enzymes, is reacted with hemicellulose and/orcellulose to obtain soluble oligosaccharides. In the first step, besidesthe enzymes from the first group, enzymes from the second group can bepresent in low activities.

In the second step, the enzyme or mixture of enzymes comprises of atleast one enzyme from the second group of enzymes, with or without oneor more of the enzymes from the first group and/or any accessory orauxiliary enzymes, and acts on reaction mixture obtained from the firststep, in the same or different reactor of any type, to obtain thefermentable sugars.

One of the embodiments of invention provides a process of hydrolysis ofhemicellulose and/or cellulose and which comprises stepwise action ofthe enzymes. The two step action minimizes inhibitory effect of the bothintermediate and final products on enzymes acting in the both steps,namely inhibitory effect of cellobiose, xylobiose and monosugars on oneor more components of cellulase and/or hemicellulase enzymes and, inparticular, on endo-glucanases, and cellobiohydrolases andxylobiohydrolases. In the present method, all steps of reaction arecarried out in the range of the pH which is favourable to the enzymes,or any mixtures thereof, more suitable results found in the range of pH4 to 8. The reaction pH in the two steps varies within the indicatedlimits depending upon the source of enzymes and the same may easilydetermined by all those skilled in the art.

In another embodiment of the present invention, the temperature of thereaction is in the range of 30° C. to 90° C., and that the operatingtemperature for a mixture of enzymes depends on the activity andstability profile of the enzymes and may be determined easily by allthose skilled in the art. The overall enzymatic hydrolysis is carriedout until all hemicellulos and/or cellulose is converted to fermentablesugars.

In accordance with the present invention in one embodiment there isprovided a process of production of fermentable sugars fromhemicellulose and/or cellulose using multi-step multi-enzyme system,wherein the process comprises treating hemicellulose and/or cellulosewith at least one enzyme of first group of enzymes at a temperatureranging from 30° C. to 90° C. to obtain a first hydrolysate, andtreating the hydrolysate obtained with at least one enzyme from secondgroup of enzymes to obtain the fermentable sugars; wherein the firstgroup and second group of enzymes are capable of hydrolysing thehemicellulose and/or cellulose.

One embodiment of the present invention provides a process of productionof fermentable sugars from hemicellulose and/or cellulose usingmulti-step multi-enzyme system, wherein the hemicellulose and/orcellulose is substantially free from lignin in particular do not containmore than 10% (w/w) lignin.

In another embodiment of the present invention, there is provided theprocess of production of fermentable sugars from hemicellulose and/orcellulose using multi-step multi-enzyme system, wherein the processcomprises treating hemicellulose and/or cellulose with at least oneenzyme of first group of enzymes at a temperature ranging from 30° C. to90° C. to obtain a first hydrolysate, and treating the hydrolysateobtained with at least one enzyme from second group of enzymes to obtainthe fermentable sugars; wherein the first group and second group ofenzymes are capable of hydrolysing the hemicellulose and/or cellulose,wherein the first group of enzymes are endo-glucanases, exo-glucanases,endo-xylanases, exo-xylanases, mannanases and galactanases.

In another embodiment of the present invention, there is provided theprocess of production of fermentable sugars from hemicellulose and/orcellulose using multi-step multi-enzyme system, wherein the processcomprises treating hemicellulose and/or cellulose with at least oneenzyme of first group of enzymes at a temperature ranging from 30° C. to90° C. to obtain a first hydrolysate, and treating the hydrolysateobtained with at least one enzyme from second group of enzymes to obtainthe fermentable sugars; wherein the first group and second group ofenzymes are capable of hydrolysing the hemicellulose and/or cellulose,wherein the second group of enzymes are xylosidases, mannosidases andglucosidases.

In yet another embodiment of the present invention, there is providedthe process of production of fermentable sugars from hemicelluloseand/or cellulose using multi-step multi-enzyme system, wherein theprocess comprises treating hemicellulose and/or cellulose with at leastone enzyme of first group of enzymes at a temperature ranging from 30°C. to 90° C. to obtain a first hydrolysate, and treating the hydrolysateobtained with at least one enzyme from second group of enzymes to obtainthe fermentable sugars; wherein the first group and second group ofenzymes are capable of hydrolysing the hemicellulose and/or cellulose,wherein optionally the enzymes are cross-linked with one or moreproteins, one or more polymers, or combinations thereof using one ormore cross linking agents.

In yet another embodiment of the present invention there is providedprotein for cross-linking of enzymes, wherein the protein is selectedfrom a group consisting of first group of enzymes, second group ofenzymes, transferrin, globulins, animal serum albumin, soy protein, wheyprotein and wheat gluten, or any combinations thereof.

In one embodiment, the present invention provides cross-linking agentsselected from a group consisting of glutaraldehyde, divinylsulphone,polyethyleneimine, and 1,4-butanedioldiglycidylether.

In still yet another embodiment of the present invention, there isprovided the process of production of fermentable sugars fromhemicellulose and/or cellulose using multi-step multi-enzyme system,wherein the process comprises treating hemicellulose and/or cellulosewith at least one enzyme of first group of enzymes at a temperatureranging from 30° C. to 90° C. to obtain a first hydrolysate, andtreating the hydrolysate obtained with at least one enzyme from secondgroup of enzymes to obtain the fermentable sugars; wherein the firstgroup and second group of enzymes are capable of hydrolysing thehemicellulose and/or cellulose, wherein the hemicellulose and/orcellulose converts into the fermentable sugars in batch process in 4 to8 hours.

In further embodiment of the present invention, there is provided theprocess of production of fermentable sugars from hemicellulose and/orcellulose using multi-step multi-enzyme system, wherein the processcomprises treating hemicellulose and/or cellulose with at least oneenzyme of first group of enzymes at a temperature ranging from 30° C. to90° C. to obtain a first hydrolysate, and treating the hydrolysateobtained with at least one enzyme from second group of enzymes to obtainthe fermentable sugars; wherein the first group and second group ofenzymes are capable of hydrolysing the hemicellulose and/or cellulose,wherein the hemicellulose and/or cellulose converts into the fermentablesugars in continuous process with hydraulic retention time of 1 to 4hours.

One embodiment of the present invention provides the fermentable sugarscomprising soluble oligosaccharides, cellobiose, glucose, xylobiose,xylose and arabinose.

Another embodiment of the present invention provides a process forobtaining hemicelluloses and/or cellulose from biomass, wherein processcomprises mixing the biomass with 5% to 10% (w/v) alkali having pH inthe range of 12-14 at a temperature ranging from 50° C. to 200° C. under1.0 to 20 bar pressure for 5 minutes to 2 hours to obtain a biomassslurry; filtering the biomass slurry to obtain filtrate comprisinghemicelluloses and residue comprising cellulose; treating the filtratewith alcohol to obtain a precipitate containing hemicelluloses; washingthe residue containing cellulose with water to remove residual alkali toobtain cellulose; and washing the precipitate to obtain hemicelluloses.

Yet another embodiment of the present invention provides a process forobtaining hemicelluloses and/or cellulosed from biomass, wherein thebiomass is selected from a group consisting of grasses, rice straw,wheat straw, cotton stalk, castor stalk, sugarcane or sorghum bagasse,corn cobs, corn stover, stalks, switch grass and elephant grass.

Another embodiment of the present invention provides a process forobtaining hemicelluloses and/or cellulosed from biomass using alkali,wherein the ratio of alkali to biomass is 0.5 to 2.0, preferably 1.4.

Another embodiment of the present invention provides a process forobtaining hemicelluloses and/or cellulose from biomass, wherein processcomprises mixing the biomass with 5% to 10% (w/v) alkali having pH inthe range of 12-14 at a temperature ranging from 50° C. to 200° C. under1.0 bar pressure for 2 hours to obtain a biomass slurry; filtering thebiomass slurry to obtain filtrate comprising hemicelluloses and residuecomprising cellulose; treating the filtrate with alcohol to obtain aprecipitate containing hemicelluloses; washing the residue containingcellulose with water to remove residual alkali to obtain cellulose; andwashing the precipitate to obtain hemicelluloses.

Another embodiment of the present invention provides a process ofobtaining hemicelluloses and/or cellulose from biomass, wherein processcomprises mixing the biomass with 5% to 10% (w/v) alkali having pH inthe range of 12-14 at a temperature ranging from 50° C. to 200° C. under1.0 to 20 bar pressure for 5 minutes to 2 hours to obtain a biomassslurry; filtering the biomass slurry to obtain filtrate comprisinghemicelluloses and residue comprising cellulose; treating the filtratewith alcohol to obtain a precipitate containing hemicelluloses; washingthe residue containing cellulose with water to remove residual alkali toobtain cellulose; and washing the precipitate to obtain hemicelluloses,wherein at least 85% hemicellulose is recovered.

Another embodiment of the present invention provides a process ofobtaining hemicelluloses and/or cellulose from biomass, wherein processcomprises mixing the biomass with 5% to 10% (w/v) alkali having pH inthe range of 12-14 at a temperature ranging from 50° C. to 200° C. under1.0 to 20 bar pressure for 5 minutes to 2 hours to obtain a biomassslurry; filtering the biomass slurry to obtain filtrate comprisinghemicelluloses and residue comprising cellulose; treating the filtratewith alcohol to obtain a precipitate containing hemicelluloses; washingthe residue containing cellulose with water to remove residual alkali toobtain cellulose; and washing the precipitate to obtain hemicelluloses,wherein at least 90% cellulose is recovered.

Another embodiment of the present invention provides a process ofproduction of fermentable sugars from biomass using multi-stepmulti-enzyme system, wherein the process comprises

-   -   a. mixing biomass with 5% to 10% w/v alkali having pH in the        range of 12-14 at a temperature ranging from 50° C. to 200° C.        under 1.0 to 20 bar pressure for 5 minutes to 2 hrs to obtain a        biomass slurry;    -   b. filtering said biomass slurry to obtain filtrate comprising        hemicellulose; and residue comprising cellulose;    -   c. treating the filtrate with alcohol to obtain a precipitate        containing hemicelluloses;    -   d. washing the residue from step (b) with water to remove        residual alkali to obtain cellulose;    -   e. washing the precipitate to obtain hemicelluloses;    -   f. treating the hemicellulose from step (e) and/or the cellulose        from step (d) with at least one enzyme of first group of enzymes        at a temperature ranging from 30° C. to 90° C. to obtain a        hydrolysate; and    -   g. treating the hydrolysate of step (f) with at least one enzyme        from second group of enzymes to obtain the fermentable sugars    -    wherein the first group and second group of enzymes are capable        of hydrolysing the hemicellulose and/or cellulose

Further embodiment of the present invention provides a process ofproduction of fermentable sugars from biomass using multi-stepmulti-enzyme system, wherein the process comprises

-   -   a. mixing biomass with 5% to 10% w/v alkali having pH in the        range of 12-14 at a temperature ranging from 50° C. to 200° C.        under 1.0 to 20 bar pressure for 5 minutes to 2 hrs to obtain a        biomass slurry;    -   b. filtering said biomass slurry to obtain filtrate comprising        hemicellulose; and residue comprising cellulose;    -   c. treating the filtrate with alcohol to obtain a precipitate        containing hemicelluloses;    -   d. washing the residue from step (b) with water to remove        residual alkali to obtain cellulose;    -   e. washing the precipitate to obtain hemicelluloses;    -   f. treating the hemicellulose from step (e) and/or the cellulose        from step (d) with at least one enzyme of first group of enzymes        at a temperature ranging from 30° C. to 90° C. to obtain a        hydrolysate; and    -   g. treating the hydrolysate of step (f) with at least one enzyme        from second group of enzymes to obtain the fermentable sugars    -    wherein the first group and second group of enzymes are capable        of hydrolysing the hemicellulose and/or cellulose; wherein the        first group and second group of enzymes are cross-linked with a        protein or a polymer using a cross-linking agent.

In another embodiment of the present invention the enzymes are recycledand reused to provide a cost effective process in terms of cost of theenzyme used per unit of hemicellulose and/or cellulose hydrolyzed tofermentable sugars. For example, the enzymes can be used in packed,stirred, or fluidized bed reactors in immobilized form, or in membranereactors, or combinations thereof.

An immobilized enzyme is an enzyme which is attached to an inert,insoluble, porous or non-porous, material. This can provide increasedstability and resistance of the enzymes to changes in conditions such asshear, pressure, pH or temperature. Immobilization also allows enzymesto be held in place, or in the confines of the reactor throughout thereaction, following which they are easily separated from the productsand may be used again.

Immobilized enzymes are cost effective as well as simple to use in morethan one cycle. The immobilized enzyme is easily removed from thereaction making it easy to recycle the biocatalyst. Immobilized enzymestypically have greater thermal and operational stability than thesoluble form of the enzyme. Immobilized enzymes can be prepared bydifferent methods. A widely used method is adsorption of the enzymes ona suitable solid porous matrix. Enzyme is attached to the solid surfaceof the matrix by a variety of methods ranging from simple adsorption tocovalent reaction. The enzyme can also be trapped in insoluble beads ormicrospheres, such as calcium alginate beads. However, these insolublesubstances hinder the arrival of the substrate, and the exit ofproducts, especially when the substrate is polymeric and bulky molecule.

The enzyme can also be covalently bonded to a matrix or anyenzyme/protein through a chemical reaction. This method is by far themost effective method among those listed here. As the chemical reactionensures that the binding site does not cover the enzyme's active site,the activity of the enzyme is only affected by immobility. The enzymeand the matrix are cross-linked through a cross-linking agent suchglutaraldehyde or carbodiimide.

According to one of the preferred embodiments of the current invention,the enzyme is immobilized on a suitable solid support. The carriers ormatrix used for immobilization may comprise of any natural or syntheticand organic or inorganic material e.g. hydrophilic synthetic polymersuch as polyacrylamides, polymethacrylamides, polyacrylates,polymethacrylates, polyimides, polyvinyl hydrophilic polymers,polystyrene, polysulfone or the like and natural or syntheticpolysaccharides such as starch, dextran, chitin agar or agarose;inorganic material such as silicious materials such as silicon dioxideincluding amorphous silica and quartz, controlled pore glass, titaniumdioxide and ceramics or suitable combination thereof.

In another embodiment of the present invention, the enzyme/s can becross-linked with itself or any other protein, or any other monomer orpolymer, by means of a cross-linking agent, to form soluble or insolubleaggregates called cross-linked enzyme aggregates (CLEA), and that can beused as immobilized enzyme, or in membrane reactors wherein, themembranes are able to retain the enzymes or enzyme aggregates as well aspolymeric substrates, while permitting smaller reaction products topermeate or pass through.

In another embodiment of the invention, when using enzymes to act uponsolid substrate, like cellulose in solid form the process cost isrendered cost effective through the recycling of the enzyme/s, theenzyme/s being used in membrane reactors as macromolecules in theirnative form, or cross-linked to itself, or to a suitable protein/enzyme,or any other monomer or polymer, using a suitable cross-linking agent toobtain active cross-linked soluble enzyme preparations with 100%membrane rejection coefficients. The proteins used for cross-linking aretransferrin, globulins, animal serum albumins, soy protein, wheyprotein, and wheat gluten.

According to one of the preferred embodiment of the present invention,the saccharification process, for cellulose and hemicellulose,separately or combined, is carried out in bioreactors and thebioreactors used for the First step and/or Second step can be packed bedor fluidized bed bioreactors, or stirred tank bioreactors that arecoupled with membrane filtration systems using micro-filtration,ultra-filtration membranes, and/or nano-filtration membranes.

According to another preferred embodiment of the current invention,hemicellulose is treated with at least one of the enzymes of the firstgroup of enzyme, wherein the enzyme/s breaks down the polymericstructure of hemicellulose to soluble oligosaccharides. Further, in thesecond step these oligosaccharides are treated with at least one of theenzymes from the second group, wherein the enzyme/s convert solubleoligosaccharides to fermentable sugars.

According to another embodiment of the present invention, the first stepof saccharification of hemicelluloses is carried out in a stirred tankbioreactor which is coupled with a membrane filtration system, such asmicrofiltration, ultrafiltration and/or nanofiltration, preferablyultrafiltration membrane alone, which retains and recycles solubleenzyme/s of the first step to the tank bioreactor, and solubleoligosaccharides pass through it. The second step is carried out in thesecond stirred tank bioreactor, which is coupled with yet anothermembrane filtration system, such as ultrafiltration or nanofiltration,preferably nano-filtration membrane which retains and recycle solubleenzyme/s of the second step as well as the larger solubleoligosaccharides while fermentable sugars will pass through as permeate.

According to another embodiment of the present invention, the First stepof saccharification of hemicelluloses is carried out in a packed bedreactor wherein the enzyme/s used in the first step are immobilized on asuitable matrix. Soluble oligosaccharides are formed in the reactor thatgoes to the second step. The Second step is carried out in the secondpacked bed reactor column containing immobilized enzymes from the secondgroup of enzymes to obtain fermentable sugars. Alternatively, the secondreactor is a stirred tank bioreactor, which is coupled with a membranefiltration system, such as ultrafiltration or nanofiltration, preferablyultrafiltration, which retains and recycles soluble enzyme/s of thesecond group as well as large oligosaccharides while fermentable sugarsare obtained as permeate.

According to another embodiment of the present invention, the First stepof saccharification of hemicelluloses is carried out in a bioreactor,which is coupled with a membrane filtration, such as ultrafiltration ornanofiltration, preferably ultrafiltration, which will retain solubleenzyme/s used in the first step and also the larger soluble andinsoluble oligosaccharides. Soluble oligosaccharides pass through themembrane, and in the second step contacted with immobilized enzyme/s ofthe second group in a packed bed reactor and converted to fermentablesugars.

According to another embodiment of the present invention, the First stepof saccharification of hemicelluloses is carried out in a packed bedreactor wherein the enzyme/s used in the first step are immobilized on asuitable matrix. Soluble oligosaccharides are formed and passed througha second column/bioreactor, wherein the enzyme/s of the Second group areimmobilized on a suitable matrix. The stream emerging from the secondcolumn reactor contains fermentable sugars.

According to another preferred embodiment of the current invention,cellulose, is treated in the First step with at least one of the enzymesof the first group of enzyme and/or some enzymes from the second group,wherein the enzyme/s breaks down basic polymeric structure of celluloseto oligosaccharides. Further in the Second step these oligosaccharidesare treated with at least one of the enzymes from the second groupand/or some enzymes from the other group, and wherein these enzyme/sconvert oligosaccharides to fermentable sugars.

One of the embodiments of invention is that oligosaccharides formedduring saccharification of cellulose, in particular cellobiose, have aninhibitory effect on the enzymes and, in particular, on endo-gluconasesand cellobiohydrolases. Further, glucosidases convert cellobiose toglucose which can also inhibit glucanases. Such inhibitory effect can beminimized by the two step treatment of the enzymes.

In another embodiment of the present invention, the enzyme/s of thefirst step can be cross-linked with the high molecular weight protein,or any other monomer, or polymer, by means of a cross-linking agent.Cellulose is an insoluble solid and hence it is unlikely that theimmobilization of the enzymes would help the enzyme action. Thus to makethe process of saccharification of cellulose cost effective through therecycling of the enzyme of the first step, first group of enzymes arecross-linked to a protein or polymer by a suitable cross-linking agent.

In another embodiment of the present invention, the first group ofenzyme/s used for saccharification of cellulose is cross-linked with thesame enzyme/s or proteins such as transferrin, globulins, animal serumalbumin, soy protein, whey protein, or wheat gluten.

In another embodiment of the present invention, the cross-linking agentused are from a group consisting of glutaraldehyde, divinylsulphone,polyethyleneimine, and 1,4-butanedioldiglycidylether.

In one of the preferred embodiment of the present invention, the Firststep of saccharification of cellulose is carried out in a stirred tankreactor which is coupled with the membrane separation assembly to retainand recycle the soluble/cross-linked enzyme/s used in the first step.The membrane separation assembly may include microfiltration,ultrafiltration or nanofiltration membranes, to retain enzymes and sugarpolymers, while soluble oligosaccharides pass through the membranes andare sent through the second reactor containing enzyme from the secondgroup. This second stirred tank reactor is also coupled with a membraneseparation assembly which may include ultrafiltration membranes ornanofiltration membranes, that retain enzymes and large oligosaccharideswhile smaller fermentable sugars pass through the membranes.

In another embodiment of the present invention, the First step ofsaccharification of cellulose is carried out in a bioreactor which iscoupled with the membrane separation assembly to retain the solublenative or cross-linked enzyme/s from the first group. The membraneseparation assembly may include ultrafiltration membranes ornanofiltration membranes, to retain and recycle enzymes and largeroligosaccharides while smaller oligosaccharides pass through thesemembranes and are sent through the second column reactor. The Secondstep is carried out in the second column or bioreactor, wherein theenzyme/s of the second step is immobilized on a suitable matrix. Theseenzymes convert soluble oligosaccharides to fermentable sugars.

According to another preferred embodiment of the current invention, amixture of hemicellulose and cellulose, is treated in the First stepwith at least one of the enzymes of the first group of enzyme and/orsome enzymes from the second group, wherein the enzyme/s breaks downbasic polymeric structure of hemicellulose and cellulose tooligosaccharides. Further in the Second step these oligosaccharides aretreated with at least one of the enzymes from the second group and/orsome enzymes from the other group, and wherein these enzyme/s convertoligosaccharides to fermentable sugars.

It is often required to conduct simultaneous saccharification ofhemicellulose and cellulose in order to obtain a combined hydrolysate ina single two step enzyme reactor assembly for subsequent single step andcombined fermentation of sugars obtained to desired products likeethanol. In such cases, the hemicellulose and cellulose obtained frombiomass are processed as a mixture for hydrolysis and saccharificationsteps. The basic logic behind the two step enzyme reaction of thepresent invention however, is found to be as applicable to the mixtureof hemicellulose and cellulose as to each single component.

One of the embodiments of invention is that oligosaccharides formedduring saccharification of hemicellulose and cellulose, in particularcellobiose and xylobiose, have an inhibitory effect on the enzymes and,in particular, on endo-glucanases and biohydrolases. Further,glycosidases convert bioses to monosugars and these can also inhibitglucanases. Such inhibitory effect can be minimized by the two steptreatment of the enzymes.

In another embodiment of the present invention, the enzyme/s of thefirst step can be cross-linked with the high molecular weight protein,or any other monomer, or polymer, by means of a cross-linking agent.Cellulose is an insoluble solid and hence it is unlikely that theimmobilization of the enzymes would help the enzyme action. Thus to makethe process of saccharification of a mixture of hemicellulose andcellulose cost effective through the recycling of the enzyme of thefirst step, the first group of enzymes are cross-linked to a protein orpolymer by a suitable cross-linking agent.

In another embodiment of the present invention, the first group ofenzyme/s used for saccharification of the mixture of hemicellulose andcellulose is cross-linked with the same enzyme/s or proteins such astransferrin, globulins, animal serum albumin, soy protein, whey protein,or wheat gluten.

In another embodiment of the present invention, the cross-linking agentused are from a group consisting of glutaraldehyde, divinylsulphone,polyethyleneimine, and 1,4-butanedioldiglycidylether.

In one of the preferred embodiment of the present invention, the Firststep of saccharification of mixture of hemicellulose and cellulose iscarried out in a stirred tank reactor which is coupled with the membraneseparation assembly to retain and recycle the soluble or cross-linkedenzyme/s used in the first step. The membrane separation assembly mayinclude microfiltration, ultrafiltration or nanofiltration membranes, toretain enzymes and sugar polymers, while soluble oligosaccharides passthrough the membranes and are sent through the second stirred tankreactor containing enzyme from the second group. This second reactor isalso coupled with a membrane separation assembly which includesultrafiltration membranes or nanofiltration membranes that retainenzymes and large oligosaccharides while smaller fermentable sugars passthrough the membranes.

In another embodiment of the present invention, the First step ofsaccharification of a mixture of hemicellulose and cellulose is carriedout in a stirred tank reactor which is coupled with the membraneseparation assembly to retain the soluble native or cross-linkedenzyme/s from the first group. The membrane separation assembly includesultrafiltration membranes or nanofiltration membranes, to retain andrecycle enzymes and larger oligosaccharides while smalleroligosaccharides pass through these membranes and are sent through thesecond column reactor. The Second step is carried out in the secondcolumn reactor, wherein the enzyme/s of the second step is immobilizedon a suitable matrix. These enzymes convert soluble oligosaccharides tofermentable small sugars.

The reactor assemblies used and described above for carrying out theprocess according to the present invention are varied to meet anyparticular requirements. Thus, the hydrodynamics of the reaction ismaintained to ensure optimal conversion of product solution by laminarflow and by keeping minimal shear in the stirred, membrane and packedreactors.

The following examples are given by the way of illustration of theinvention contained in the present invention and therefore should not beconstrued to limit the scope of the present invention.

EXAMPLES

It should be understood that the following examples described herein arefor illustrative purposes only and that various modifications or changesin light of the specification will be suggestive to person skilled inthe art and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

Example 1 Preparation of Hemicelluloses from Cotton Stalks

3 gm of dried and size reduced cotton stalk is treated with 100 ml of 5%alkali at 121° C. for 30 minutes in an autoclave or pressure reactor.The treated sample is filtered to remove the solid residue. The filtratecontaining hemicelluloses is treated with 500 ml absolute ethanol. Theprecipitate obtained was filtered and washed with excess ethanol toobtain hemicellulose as a buff colored powder.

Example 2 Preparation of Cellulose and Hemicelluloses+Cellulose Mixturefrom Rice Straw

1 gm of dried and size reduced cotton stalk is treated is 20 ml of 5%alkali at 121° C. for 15 minutes in an autoclave. The treated sample isfiltered and washed with water to recover the solid residue as cellulosefraction. The filtrate containing hemicellulose is treated with 50 mlabsolute ethanol, and the precipitate obtained is filtered and washedwith excess ethanol to obtain hemicellulose as a buff coloredprecipitate. The cellulose residue and hemicellulose so obtained aremixed to give mixture of hemicellulose and cellulose for furtherprocessing.

Example 3 Hydrolysis of Hemicellulose

(a) Batch Reaction without Enzyme Recycle

Hemicellulose (40 g) in 1000 mL of 50 mM citrate buffer pH 5 was treatedwith 4000 IU of a pre-dominantly endo-xylanase enzyme at 50° C. for 2hours. One unit of enzyme is defined as the micromoles of product givenby one milliliter of enzyme per minute. Gel permeation Chromatographyshowed that xylobiose was the major product. In the second step 2000 IUof xylosidase enzyme was used as the second group of enzymes and addedto the reaction medium. Complete conversion of hemicellulose to xylosewas obtained over within next 2.0 hours. Table 1 gives the details ofthe reaction process with HM1 being the first enzyme and HMII being thesecond enzyme.

(b) Continuous Hemicellulose Hydrolysis with Enzyme Recycle

A 3% w/v suspension of hemicellulose in 50 mM citrate buffer pH 5.0 wasadded at about 10 mL/min using a metering pump so as to maintain theliquid level at a constant height in a 500 mL jacket heated stirredreactor wherein a total of 1000 IU of endo-xylanase was also added inone lot initially. The stirred reactor was maintained at 50° C. andcoupled with a membrane filtration system. The entire reactor assemblyconsisted of a stirred tank reactor (500 mL) equipped with a peristalticpump that circulated the reaction mass through a tubular ultrafiltrationmembrane system (5 KDa and 0.01 square meter). The retentate from themembrane system was sent back to the stirred tank while the permeate wascollected in beaker from which another peristaltic pump passed thereaction mass through a packed bed of immobilized beta-xylosidase (10 mmdia×500 mm H). The flow rate of the permeate and column was maintainedat 10 mL/min. The flow from the second reactor was analysed for glucosecontent. 95% conversion of hemicellulose to monosugars was found tooccur on continuous steady state basis. An overall average hydraulicretention time of 40-50 minutes was maintained.

Example 4 Hydrolysis of Mixture of Hemicellulose and Cellulose

(a) Batch Reaction without Enzyme Recycle

The mixture of hemicellulose and cellulose (40 g) was suspended inacidified water (pH 5) and was treated with a 4000 IU of mixture ofendo- and exo-glucanases at 50° C. for 2.0 hour. One unit of enzyme isdefined as micromoles of glucose equivalent reducing sugars produced permilliliter of enzyme per minute. Gel permeation chromatography ofreaction mixture after 2 hours showed that soluble oligosaccharides arethe major products formed. The second step reaction was carried outusing a mixture of beta-glucosidase and beta-xylosidase (each 1000 IU)was added to the reaction mixture from the first step. Completeconversion of polysaccharides to glucose and xylose was obtained withinnext 2.0 hours. The results of the reaction progress are given in Table2.

(b) Continuous Reaction with Enzyme Recycle

A 4% w/v suspension of a mixture of hemicellulose and cellulose (in aratio of 3:7) in acidified water (pH 5) was added at about 15 mL/minusing a metering pump so as to maintain the liquid level at a constantheight in a 500 mL jacket heated stirred reactor to which was added inone lot 1000 IU of mixture of endo- and exo-glucanases. The stirred tanktemperature was maintained at 50° C. The membrane reactor assembly wassame as the one mentioned in Example 3(b). The stirred reactor wascoupled with a membrane filtration system. The retentate from themembrane system was sent back to the stirred tank while the permeate wascollected in beaker from which another peristaltic pump passed thereaction mass through a packed bed of mixture of immobilizedbeta-glucosidase and immobilized beta-xylosidase (10 mm diameter×500 mmH). The flow rate of the permeate and column was maintained at 15mL/min. The flow from the second reactor was analysed for glucosecontent. An average of 90% combined conversion to monosugars was foundto occur on continuous steady state basis. An overall average hydraulicretention time of 40-50 minutes was maintained.

Example 5 Hydrolysis of Cellulose (a) Batch Hydrolysis of Cellulose

Cellulose (10 g) was suspended in 50 mM citrate buffer pH 4.8 and wastreated with 1000 IU of mixture of endo- and exo-cellulases in a stirredreactor at 50° C. One unit of enzyme is defined as the micromoles ofproduct given by one milliliter of enzyme per minute. The GPC showedthat oligosaccharides are major products after 2.0 hrs. Then in thesecond step, beta-glucosidase (500 IU) was added to reaction medium.Complete conversion of cellobiose to glucose is over within next 2.0hours.

(b) Continuous Hydrolysis of Cellulose with Enzyme Recycle

A 3% w/v suspension of cellulose in 50 mM citrate buffer pH 5.0 wasadded at about 10 mL/min using a metering pump so as to maintain theliquid level at a constant height in a 500 mL jacket heated stirredreactor wherein a total of 1000 IU of endo-xylanase was also added inone lot initially. The stirred reactor was coupled with a membranefiltration system. A one lot addition of 1000 IU of mixture of endo- andexo-cellulases was added to the stirred tank and the reaction conductedat 50° C. The reactor assembly consisted of a stirred tank reactorequipped with a peristaltic pump that circulated the reaction massthrough a tubular ultrafiltration membrane system (5 KDa and 0.01 squaremeters). The retentate from the membrane system was sent back to thestirred tank while the permeate was collected in beaker from whichanother peristaltic pump passed the reaction mass through a packed bedof immobilized beta-glucosidase (10 mm diameter×500 mm H). The flow rateof the permeate and column was maintained at 15 mL/min. The flow fromthe second reactor was analysed for glucose content. 90% conversion ofcellulose to glucose was found to occur on continuous steady statebasis. An overall average hydraulic retention time of 40-50 minutes wasmaintained.

Example 6 Preparation of Crosslinked Cellulase

The mixture of exo-glucanase and/or endo-glucanase having a totalactivity of 10 IU was crosslinked with soy protein isolate (2 mg/ml)prepared in the laboratory using glutaraldehyde as cross-linking agentunder alkaline conditions. The cross-linking reaction period wascontrolled to obtain soluble preparation enzyme aggregates (as evidencedon native PAGE). The preparation was diafiltered and concentrated on a30 KDa ultrafiltration membrane in order to remove non-cross-linkedproteins and excess cross-linking agent. The liquid preparationcontaining 50 mg/mL protein was used as the enzyme preparation.

Example 7 Continuous Hydrolysis of Cellulose Using Cross-LinkedCellulase

A 3% w/v suspension of pure cellulose in 50 mM citrate buffer pH 5.0 wasadded at about 10 mL/min using a metering pump so as to maintain theliquid level at a constant height in a 500 mL jacket heated stirredreactor wherein a total of 1000 IU of endo-xylanase was also added inone lot initially. The stirred reactor was coupled with a membranefiltration system. One lot of 1000 IU equivalent of cross-linkedexoglucanase and endoglucanase activity was added to the stirred tankwhich was maintained at 45° C. The reaction mixture was continuouslyrecirculated through a 30 KDa ultrafiltration membrane. The permeate waspassed through a packed bed column containing immobilized enzyme as usedin Example 5(b). The reaction mixture from the packed column reactor wasanalysed for glucose content. An average conversion of about 90%conversion of cellulose to glucose was found to occur on continuoussteady state. An overall average hydraulic retention time of 40-50minutes was maintained.

TABLE 1 Comparison of the rate and extent of enzymatic hydrolysis ofhemicellulose using two hemicellulase enzyme preparations HMI and HMII,each predominantly containing enzymes from the first group and thesecond group, respectively. Experiment 1 is the traditional case wherethe two enzyme preparations were used together, and Experiments 2 and 3are where the two enzyme preparations have been used in two steps asdescribed in the present invention. Saccharification percentage in hoursEnzymes 1 2 3 4 24 Experi- HM I + HM II at ND 70 ND 80 100 ment 1 startExperi- HM I (1 hr) followed 37 78 84 86 100 ment 2 by HM II Experi- HMI (2 hr) followed ND 56 84 100 — ment 3 by HM II

TABLE 2 Comparison of the rate and extent of enzymatic hydrolysis ofcellulose + hemicellulose in the ratio 64:20 using two cellulase enzymepreparations cellulase and cellulase + glucosidase, each predominantlycontaining enzymes from the first group and the second group,respectively. Experiment 1 is the traditional case where the two enzymepreparations were used together, and Experiments 2 is where the twoenzyme preparations have been used in two steps as described in thepresent invention. Percentage Saccharification Reaction Experiment 1:Experiment 2: time (hrs) Cellulase Cellulase + Glucosidase 1 60.5 62.0 263.7 81.0 4 68.8 96.4 6 71.6 98.1

1-19. (canceled)
 20. A process of production of fermentable sugars froma mixture of hemicellulose and cellulose using a multi-step multi-enzymesystem, the process comprising: a. treating said mixture ofhemicellulose and cellulose with at least one enzyme selected from agroup consisting of endo-glucanases, exo-glucanases, endo-xylanases,exo-xylanases, mannanases and galactanases at a temperature ranging from30° C. to 90° C. to obtain a hydrolysate; b. separating the hydrolysatefrom the at least one enzyme used in step (a) to obtain a solutioncomprising oligosaccharides, disaccharides, and monosugars; and c.treating the solution with at least one enzyme selected from a groupconsisting of xylosidases, mannosidases and glucosidases to obtain thefermentable sugars.
 21. The process of claim 20, wherein thehemicellulose and/or cellulose do not contain more than 10% (w/w)lignin.
 22. The process of claim 20, wherein the enzymes arecross-linked with one or more proteins, one or more polymers, orcombinations thereof using one or more cross linking agents.
 23. Theprocess of claim 22, wherein the protein is selected from a groupconsisting of first group of enzymes consisting of endo-glucanases,exo-glucanases, endo-xylanases, exo-xylanases, mannanases andgalactanases; second group of enzymes consisting of xylosidases,mannosidases and glucosidases; transferrin, globulins, animal serumalbumin, soy protein, whey protein and wheat gluten, or any combinationsthereof.
 24. The process of claim 22, wherein the cross linking agent isselected from a group consisting of glutaraldehyde, divinylsulphone,polyethyleneimine, and 1,4-butanedioldiglycidylether.
 25. The process ofclaim 20, wherein the mixture of hemicellulose and cellulose convertsinto the fermentable sugars in batch process in 4 to 8 hours.
 26. Theprocess of claim 20, wherein the mixture of hemicellulose and celluloseconverts into the fermentable sugars in continuous process withhydraulic retention time of 1 to 4 hours.
 27. The process of claim 20,wherein the fermentable sugars comprise soluble oligosaccharides,cellobiose, glucose, xylobiose, xylose and arabinose.
 28. The process ofclaim 20, wherein the mixture of hemicellulose and cellulose is obtainedby a process comprising a. mixing biomass with 5% to 10% w/v alkalihaving pH in the range of 12-14 at a temperature ranging from 50° C. to200° C. under 1.0 to 20 bar pressure for 5 minutes to 2 hrs to obtain abiomass slurry; b. filtering said biomass slurry to obtain filtratecomprising hemicellulose; and residue comprising cellulose; c. treatingthe filtrate with alcohol to obtain a precipitate containinghemicelluloses; d. washing the residue from step (b) with water toremove residual alkali to obtain cellulose; and e. washing theprecipitate to obtain hemicellulose.
 29. The process of claim 28,wherein the biomass is selected from the group consisting of grasses,rice straw, wheat straw, cotton stalk, castor stalk, sugarcane orsorghum bagasse, corn cobs and corn stover, stalks, switch grass, andelephant grass.
 30. The process of claim 28, wherein the ratio of alkalito biomass is 0.5 to 2.0.
 31. The process of claim 28, wherein saidpressure is 1.0 bar.
 32. The process of claim 28, wherein said time is 2hrs.
 33. The process of claim 28, wherein at least 85% hemicellulose isrecovered.
 34. The process of claim 28, wherein at least 90% celluloseis recovered.
 35. A process of production of fermentable sugars frombiomass using multi-step multi-enzyme system, the process comprising: a.mixing biomass with 5% to 10% w/v alkali having pH in the range of 12-14at a temperature ranging from 50° C. to 200° C. under 1.0 to 20 barpressure for 5 minutes to 2 hrs to obtain a biomass slurry; b. filteringsaid biomass slurry to obtain filtrate comprising hemicellulose; andresidue comprising cellulose; c. treating the filtrate with alcohol toobtain a precipitate containing hemicelluloses; d. washing the residuefrom step (b) with water to remove residual alkali to obtain cellulose;e. washing the precipitate to obtain hemicelluloses; f. treating thehemicellulose from step (e) and the cellulose from step (d) with atleast one enzyme selected from a group consisting of endo-glucanases,exo-glucanases, endo-xylanases, exo-xylanases, mannanases andgalactanases at a temperature ranging from 30° C. to 90° C. to obtain ahydrolysate; g. separating the hydrolysate from the at least one enzymeused in step (f) to obtain a solution comprising oligosaccharides,disaccharides and monosugars; and h. treating the solution with at leastone enzyme selected from a group consisting of xylosidases, mannosidasesand glucosidases to obtain the fermentable sugars.